(soft music)
- [Lydia] Welcome to the Lessons from Lab and Life Podcast.
I'm your host Lydia Morrison, and I hope
that this podcast offers you some new perspective.
This is the second episode of our
COVID-19 Researcher Spotlight Series,
aimed at highlighting preprints, publications, protocols
and other research related to COVID-19,
all in 19 minutes or less.
Today, I'm joined by molecular biologist,
Feng Zhang of the Broad Institute
and his former students who now have a lab of their own,
Jonathan Gootenberg and Omar Abudayyeh
of the McGovern Institute for Brain Research at MIT.
Feng, Omar and Jonathan,
thank you so much for joining me today.
- [Feng] Thank you for having us.
- [Lydia] I was wondering if we could start by
your explaining a bit about your STOPCovid Research.
- [Feng] Sure, we started to work on STOPCovid
when we first learned about the Coronavirus pandemic.
This was back in January and I remember reading an article
in The New York Times saying that there's a new virus
that's causing havoc for the public health system in China.
And we have been working on using CRISPR proteins
for detecting nucleic acids and pathogens
before we developed a technique called SHERLOCK
and we thought maybe given this public health concern,
we can start to repurpose what we developed before
so that we can use it to detect coronavirus.
And since then we've been working on trying to make
the approach as sensitive
and also as easy to use as possible.
And that's what led to STOPCovid.
- [Lydia] So how does this STOPCovid method work?
- [Feng] Omar or Jonathan, do you want to start with that?
- [Jonathon] Yeah, this is Jonathan.
So STOPCovid relies on the basic technology
of CRISPR diagnostics, which is essentially
the use of these CRISPR enzymes
and their interesting collateral effect
to detect nucleic acids and then trigger a actual cleavage
while the nucleic acid's in solution
that allows us to produce a detectable signal.
So what happens is that when these CRISPR enzymes,
and the CRISPR enzyme we're using in STOPCovid is Cas12b,
but when it detects a sequence
that it's programmed to target
and this is a sequence that corresponds
to a place in the SARS-CoV-2 genome,
it becomes activated and actually will then cut a reporter
that we've doped into the solution,
which can produce a fluorescence.
One of the interesting things about STOPCovid
is that we've coupled in the same solution
this CRISPR detection with an isothermal amplification.
So that is an amplification similar in principle to PCR
where we're getting exponential production of a DNA species,
but it occurs at a single temperature.
So we don't have to go through any cycling steps.
So that amplification, which is called LAMP,
occurs at 60 degrees Celsius or 60 to 65 degrees Celsius.
And we can do that in the same reaction
as this CRISPR detection.
So what essentially happens is that when we
do the LAMP amplification on the RNA genome of SARS-CoV-2,
then we can produce a lot of DNA that can then basically
be very rapidly detected by the Cas12
to produce a fluorescence or a signal,
lateral flow signal we can detect in the strip very easily.
So it's a very simplified test for these molecular
diagnostics that needs much less equipment.
- [Lydia] Less equipment sounds good,
especially for places that don't have access to large
instrumentation necessary to run like RT-PCR or sequencing.
What are the other advantages of the STOPCovid test?
- [Omar] Some of the main advantages of the STOPCovid
tests are really the price point,
as well as the portability of the test.
And so a really big thing that we thought about
when we were trying to simplify our original technology
was how to limit the number of steps there are
for the user in terms of fluid handling,
as well as points where you could open up the tubes
and cause spread of your reaction,
which can cause false positives and future reactions
that contamination actually really impacted the CDC test.
And so the ability to make this into a single reaction
with very limited steps for the user
really makes us as portable as possible
for being able to go into maybe people's homes
or point of care settings in the future.
And then of course the price is something
we've been really thinking about.
And so the components and the chemistries that go into it
are readily scalable and will help to hopefully
make this possible for daily testing
or every other day testing in the future
which is a real goal of ours.
- [Lydia] So what makes this test more scalable
than those currently available?
- [Feng] The goal of developing STOPCovid
is to make the reaction as easy to perform as possible.
And also to have a high level of sensitivity.
I think one of the advantages of STOPCovid
is that the system can all work within a single step
and the user will simply put a sample
into the STOPCovid workflow and then be able to read out
the result through this streamlined workflow process.
The reagents that go into creating a STOPCovid reaction
is not that different than other amplification techniques
like LAMP or even PCR,
is a few proteins that will have to be prepared.
And we've been getting quite a bit of help from NEB
with purifying some of these proteins,
which has been really helpful for us.
And so once you have all that,
then the ability to be able to run isothermal reaction,
but with high levels of sensitivity, I think that's what
makes STOPCovid particularly interesting.
- [Lydia] So I think Omar mentioned that this is a test
that someone might be able to do at home,
is that one of the future goals for the STOPCovid test?
That this would be a home diagnostic
like a pregnancy test that someone could take?
- [Feng] That is the ultimate goal of making the diagnostics
something that people can use in the comfort
of their own homes so that they don't have to travel
to the hospital and if they are infected with the virus,
during that trip there's the chance of them
exposing other people to the virus.
So that's the goal.
There are few things that we'll have to work out
in order to make this something that is truly
a turnkey so that people can run in their homes.
So we're working on figuring out how we might be able
to develop a device that can make the process
very streamlined and just one step
and get it resolved in a very fail proof type of way.
- [Lydia] So how would those results be read out?
How are the results delivered in the STOPCovid assay?
- [Jonathan] In our current white paper that we released,
we actually had the readout with a visual readout
where the collateral activity cleaves a DNA species
that can then be detected by a lateral flow strip,
which essentially has the same readout properties
as a pregnancy test.
It's a line, but one of the very nice things
about this technology is that it's very modular.
So we can change the readout from a lateral flow readout
to a fluorescent readout and vice versa.
So for future iterations, each of these
different readouts has different values.
Obviously the lateral flow readout has the idea
that you don't need any sort of readout instrumentation,
you can do it by eye.
But fluorescent readouts can be faster
because you can read them in real time
and that can also lead sometimes
to more quantitative readout.
So in future iterations of the chemistry
and how the technology goes,
we're envisioning either readout as potential options
and looking at the positive negatives of both.
- [Lydia] And so I'm thinking about the utility
of this as a home test and obviously a pregnancy test
is easy to take, but performing a nasopharyngeal swab
at home would obviously be a little bit more difficult
and require a little bit more training.
So what sort of samples would this test work on?
- [Omar] I think nasopharyngeal swabs
have been used heavily in the past and yeah,
they're not ideal for someone to take on their own
because I think they're pretty invasive and uncomfortable.
So, a big goal of STOPCovid was
how do we port this over to other tests?
And so there's been a number of papers
on saliva being useful and having similar viral loads
and maybe some people have made cases
that might be even higher viral loads
than other sample types.
And of course, there's also anterior nare swabs
which is basically a nasal swab.
And so we've actually shown in the white paper
that we can work from the saliva matrix
and we're exploring saliva samples
and the ultimate goal is hopefully
to move towards nasal swabs
because with saliva and nasal swabs
someone can easily provide that sample type on their own.
- [Lydia] Absolutely, you've mentioned that
isothermal amplification step,
which is great because it occurs at one temperature
and one temperature is probably something
that someone could achieve at home
with maybe not a cup of hot water,
but maybe like a sous vide type instrument.
What is the sensitivity of this test like?
And how does it compare to some of the RT-qPCR tests
that are currently available?
- [Jonathan] So isothermal amplifications
are really great because they can be read out
without having to change the temperature unlike PCR.
And they're also very rapid and very sensitive.
So in our current publication
or I should say white paper that we released,
we have a limited detection of a hundred viral copies
and we're working to push that farther
using additional methods.
One thing is that if you compare these isothermal methods
or I should say point of care methods
to traditional readouts, like qPCR.
In qPCR, the current clinical protocol involves
concentration and purification,
which while very difficult to do in a point of care setting
because it requires instrumentation
and often expert handling of the sample,
does have the value of taking potentially
a lot of volume and putting a lot more
concentrated viral sample into that.
So one thing that we're looking to do
in future iterations is potentially figure out
ways that we can increase concentration
and break the limit of what you can do
without a concentrated sample.
So that's one thing that we're thinking about.
- [Lydia] That's excellent.
So could you guys tell me how
you all got involved in this project together?
- [Feng] So Jonathan, Omar and I have been
working together many years now.
They were graduate students in my lab
and now they are independent PIs at MIT as junior fellows.
When the coronavirus issue first came up in the news,
we saw the news and realized that
this is going to be a critical problem.
And because we're having a working on
SHERLOCK diagnostics before,
we thought it would be great to develop SHERLOCK
so that we can use it to detect coronavirus.
And as we started to develop the technique,
we sent reagents out to a number of
collaborators around the world.
They have given us feedback that the reaction works well,
it's sensitive and in fact one of our collaborators
in Thailand has received approval
so that they are using it in their hospitals
to be able to screen new patients
who were coming in for surgery
so that they can triage them differently
based on their infection status.
But one of the feedback that we received
is also that the reaction is a little challenging
to do because you have to run the amplification first
and then combine it with the CRISPR detection step.
And that transfer step can pose risk
for a sample contamination.
And so given all those feedback
we then went back to the drawing board
to think about how can we overcome
these challenges and then we thought
maybe we can develop a robust one pot reaction.
And then that's when we thought maybe
integrating the PreAmp and also with the new CRISPR protein
that can be stable, and during the amplification process
will help us get there.
We've sent that reagent out
to a number of collaborators as well.
And now one of the things that we are focused on
is to make it more sensitive
because as we try to develop the test
to be broadly used, we want to make sure that
it has as low of false negatives as possible.
And that is something that we're focused on now.
Hopefully it will have more improvements to report soon.
And then simultaneously we're collaborating
with others to see how we can best
get this into a point of care
or in a simple format so that people can use it
in a much more widely distributed way.
- [Lydia] That's excellent.
And I certainly look forward to
seeing it be available for home use
certainly many more diagnostics are going to be necessary
in order for the globe really to start opening up,
opening borders more and people to get back to life
in a more natural, more normal way,
the way that we were used to.
And I really appreciate all the efforts from your group,
all of your groups, as well as the collaborations.
I think that this has been a great time
to really emphasize the scientific collaborations
that happen around the globe.
And it's really refreshing I think to see
these surrounding medical problems
and solving these global health problems.
And so hopefully this will continue into the future as well.
- [Feng] Yeah, definitely.
And I think this has really been one of the most
collaborative periods in science
that we have all experienced.
Scientists from around the world are really coming together
recognizing that we are all universally faced
with one common problem.
And then by joining our efforts together
we'll be able to get out of this sooner.
But also I want to just thank you guys,
NEB has been a great partner working with us
providing proteins and expertise,
which has been very helpful
as we are working in this challenging time
to get access to reagents as quickly as possible.
So thank you all for all of your help too.
- [Lydia] Well speaking on behalf of NEB,
I can say that it is our honor and privilege
to be able to help support your research.
And we're very happy to do so.
Just as a closing, could you tell us something good,
do any of you have any good stories
about maybe things that have happened
or reflections that you've had during
this time in quarantine?
- [Jonathon] It's been an exciting time to see,
as Feng mentioned, people come together
and be more collaborative
and in the context of all of this,
it's also still enjoyable
to see the science of this discovery process.
So the Cas12b enzyme that we're using here
is one that's from a very thermophilic bacterium,
that's actually spoils fruit juices,
which is an interesting little anecdote
about where these different enzymes come from.
So it's neat to still keep going back
and mining these different enzymes for these properties.
Similarly, during the process of this,
we've done some optimizations that are just fun to do.
We did a screening panel of different additives
to increase the activity of the reaction.
And the one that actually came out is
the best was a taurine,
which is in certain energy drinks
and it's a neat stabilizing molecule.
So it's neat to still be able to go back to the lab
and tinker around and see these cool discoveries,
even if it's in the context of everything that's going on.
So you have to find these moments of joy
in the backdrop of maybe a broader context.
- [Lydia] I'm glad that you've been able
to find some moments of joy.
I know that you've all been working very, very hard
and diligently on this research,
on this STOPCovid kit and method.
And it's wonderful to see all the results shared so quickly.
It's nice to see the white papers
and the preprints come out so regularly.
It's nice to see this equal and open sharing
of scientific information and knowledge.
And it's certainly, I think that something that
the public is really desirous of at this particular time.
So thank you all so much for your work.
- [Feng] Thank you.
- [Jonathan] Thank you for having us.
- [Lydia] Thanks so much for joining me today as well.
And hopefully we can speak again sometime.
- [Feng] Yeah, take care, stay healthy in the meantime.
- [Lydia] Thank you, you as well.
Thanks for tuning into this episode,
be sure to check out the transcript of this podcast
for helpful links to further resources
and tune in for our COVID-19 Researcher Spotlight Series
episode next week, which will actually be in an NEB TV
episode in which I'm interviewing Nathan Tanner,
a senior scientist here at NEB and our resident expert
on Loop-mediated Isothermal Amplification,
or LAMP technology.
Nathan has a great perspective on lots of different
applications of LAMP for SARS-CoV-2 diagnostics.
So join us and find out how this single temperature
amplification method is being used to
create simple and robust COVID-19 diagnostics.